Internal standards labelled samples

The wheat sample residue level is determined from the relative mass spectral responses of the analytes to the corresponding isotopically labeled internal standards. The sample relative response is compared with the average relative response of a standard solution of analyte and internal standard analyzed before and after the sample (bracketing standards). Both samples and standards receive the same amount, 100 ng, of each internal standard to facilitate the comparison. The calculations to determine the residue level in wheat tissues are outlined in Section 7.3.1. 

Soil, sediment (CDDs) Addition of isotopically labeled internal standards to sample addition of Na2S04 and extraction with hexane/methanol or Soxhlet extraction with toluene clean-up using column chromatography if needed volume reduction HRGC/MS (EI/SIM) No data No data Eschenroeder et al. 1986... 

Gas chromatography-selected ion monitoring (GC-SIM) is often used for the quantification of GAs by GC-MS, an internal standard labeled with stable isotopes (usually with deuterium) being used as the most reliable and sensitive method. A mass chromatogram reconstructed from full-scan GC-MS is also used for semiquantification. The amounts of GAs are determined by measuring the peak areas of ions characteristic for each GA and comparing these areas to those of authentic samples. When an internal standard labeled with a stable isotope is used, the ratio of the area of an ion peak characteristic for a sample and the area of the corresponding peak of the labeled internal standard is used for quantification. The analysis of GAs by GC-MS has been discussed in numerous publications and review articles.254-256... 

The PCDDs/PCDFs are quantitated by comparing the MS response of the detected analyte relative to the MS response of the appropriate 13C labeled internal standard (Table 2). The responses of both the ions monitored for each analyte are used for quantitation. The labeled internal standards are added prior to sample extraction. Thus, the quantitative results for the native analytes are corrected for the recovery of the internal standards, based on the assumption that losses of the internal standards during sample preparation and analysis are equal to the losses of the unlabeled PCDDs/PCDFs. 

Two general methods utilizing the stable isotope dilution technique have been successfully used for the quantification of drugs and their metabolites by ms. The simplest, but least sensitive and specific method, is a non-chromatographic technique that utilizes chemical ionization mass spectrometry (cims) and internal standards labeled with stable isotopes. This method has been used to quantify the antiarrhythmic drugs, lidoq lne and quinidine and some of their metabolites, in human plasma samples. 

Fig. 1. Difference gel electrophoresis (DICE) workflow. An aliquot of each sample is mixed to create the internal standard. All samples and the standard are labeled, and then combined before running the 2-D gel. Image acquisition and analysis complete the workflow. Obtained with permission from Westermeier R, Scheibe B. Difference gel electrophoresis based on lys/cys tagging. In Posch A. Ed. Sample Preparation for 2D PAGE. Methods in Molecular Biology, Humana Press, Totowa, NJ (2007).

This labeled peptide, known as an AQUA peptide, acts as an internal standard. The sample is spiked with a known amount of the AQUA peptide and is analyzed by LC-selected reaction monitoring (SRM). The different masses of the endogenous signature peptide and the AQUA peptide are resolved by SRM tandem MS analysis, and the amount of the native peptide is calculated from the known amount of the AQUA peptide and the ratio of the area under the peaks. The AQUA approach can also be combined with MALDI-MS. 

Isotope Because isotopically labeled materials are as chemically close to the analyte as possible without being identical to it, these should be the most satisfactory internal standards, fulfilling the majority of requirements. However, difficulties arise. First, suitably labeled materials have to be available or synthesized and, second, they have to be quantified. When using radiolabeled internal standards, the sample normally has to be divided for the radioactivity to be counted. Molecules containing stable isotopes are usually quantified by MS. Furthermore, there is increasing evidence that isotopic effects are more common than was once thought and assumptions of identical chemical properties caimot be made. 

Similarly, a direct multiplex assay of lysosomal enzymes in dried blood spots has been developed for newborn screening [18]. This approach is based on the incubation of dried blood spots at 37°C overnight with the appropriate substrates and stable isotopically labeled internal standards. If the enzyme was fully active, substrate was converted completely to the corresponding product which was quantified based on its relationship to the known concentration of the internal standard. Importantly, samples without dried blood spots ( blank ) have to be used to adjust for background noise. Corresponding enzyme activities were calculated based on the assumption that 10 xl of extraction solution contained 0.98 (xl of blood [18]. 

Radiochemical methods of analysis take advantage of the decay of radioactive isotopes. A direct measurement of the rate at which a radioactive isotope decays may be used to determine its concentration in a sample. For analytes that are not naturally radioactive, neutron activation often can be used to induce radioactivity. Isotope dilution, in which a radioactively labeled form of an analyte is spiked into the sample, can be used as an internal standard for quantitative work. 

Quantitative mass spectrometry, also used for pharmaceutical appHcations, involves the use of isotopicaHy labeled internal standards for method calibration and the calculation of percent recoveries (9). Maximum sensitivity is obtained when the mass spectrometer is set to monitor only a few ions, which are characteristic of the target compounds to be quantified, a procedure known as the selected ion monitoring mode (sim). When chlorinated species are to be detected, then two ions from the isotopic envelope can be monitored, and confirmation of the target compound can be based not only on the gc retention time and the mass, but on the ratio of the two ion abundances being close to the theoretically expected value. The spectrometer cycles through the ions in the shortest possible time. This avoids compromising the chromatographic resolution of the gc, because even after extraction the sample contains many compounds in addition to the analyte. To increase sensitivity, some methods use sample concentration techniques. 

It is possible to carry out a chromatographic separation, collect all, or selected, fractions and then, after removal of the majority of the volatile solvent, transfer the analyte to the mass spectrometer by using the conventional inlet (probe) for solid analytes. The direct coupling of the two techniques is advantageous in many respects, including the speed of analysis, the convenience, particularly for the analysis of multi-component mixtures, the reduced possibility of sample loss, the ability to carry out accurate quantitation using isotopically labelled internal standards, and the ability to carry out certain tasks, such as the evaluation of peak purity, which would not otherwise be possible. 

Erbs, M., Hoerger, C.C., Hartmaim, N. and Bucheli, T.D. (2007). Quantification of six phytoestrogens at the nanogram per liter level in aqueous environmental samples using C-13(3)-labeled internal standards. Journal of Agricultural and Food Chemistry 55, 8339-8345. 

It is clear that neither NMEA nor NDPA is appropriate for an internal standard in NDMA determination if criteria are interpreted strictly, but both compounds have been used for this purpose. Addition of a nitrosamine, not normally present in the sample, is helpful in detecting any gross errors in the procedure, but the addition should not be considered to be internal standardization. Utilization of NMEA or NDPA to indicate recovery of NDMA can lead to significant errors. In most reports of the application of these "internal standards", recovery of all nitrosamines was close to 100%. Under these conditions, any added compound would appear to be a good internal standard, but none is necessary. NDMA is a particularly difficult compound for use of internal standardization because of its anomalous distribution behavior. I mass j ectrometry is employed for quantitative determination, H- or N-labeled NDMA could be added as internal standard. Because the labeled material would coelute from GC columns with the unlabeled NDMA, this approach is unworkable when GC-TEA is employed or when high resolution MS selected ion monitoring is used with the equipment described above. 

Radioisotope-labeled nitrosamines have proven valuable in development of analytical methods and for demonstrating efficiency of recovery of nitrosamines from tobacco products and smoke (37-39). The very high specific activity required for low part-per-billion determinations has discouraged most analysts from using this approach. Unless a radiochromatographic detector with adequate sensitivity is available, samples must be counted independently of the final chromatographic determination, and one of the advantages of internal standardization, correction for variation in volume injected, is lost. 

The method using GC/MS with selected ion monitoring (SIM) in the electron ionization (El) mode can determine concentrations of alachlor, acetochlor, and metolachlor and other major corn herbicides in raw and finished surface water and groundwater samples. This GC/MS method eliminates interferences and provides similar sensitivity and superior specificity compared with conventional methods such as GC/ECD or GC/NPD, eliminating the need for a confirmatory method by collection of data on numerous ions simultaneously. If there are interferences with the quantitation ion, a confirmation ion is substituted for quantitation purposes. Deuterated analogs of each analyte may be used as internal standards, which compensate for matrix effects and allow for the correction of losses that occur during the analytical procedure. A known amount of the deuterium-labeled compound, which is an ideal internal standard because its chemical and physical properties are essentially identical with those of the unlabeled compound, is carried through the analytical procedure. SPE is required to concentrate the water samples before analysis to determine concentrations reliably at or below 0.05 qg (ppb) and to recover/extract the various analytes from the water samples into a suitable solvent for GC analysis. 

Analytical accuracy. The mixture of all deuterium-labeled internal standards is added to each water sample before extraction. This does not prevent the loss of the unlabeled herbicides from the sample in subsequent processing steps, but a proportional loss of the deuterated internal standard precludes the need to correct for recovery. Although referring to recovery in this type of analysis is inappropriate, the accuracy of this method should be monitored. 

Wheat samples are extracted with dilute ammonia on the ASE200. The extracts are amended with isotopically labeled internal standards. The extracts are purified by sequential octadecyl reversed-phase solid-phase extraction (Cig SPE) and ethylenediamine-iV-propyl anion exchange (PSA) SPE. The samples are analyzed by LC/MS/MS. This method determines crop residues of flucarbazone-sodium and A-desmethyl flucarbazone with a limit of quantitation (LOQ) of 0.01 mgkg for each analyte. 

Acidify a 10-g water sample with 60 pL of formic acid. The sample may be stored in a refrigerator until needed. Add the appropriate amount of Ce-labeled internal standards. (An amount of 0.25 ng of -labeled internal standard in a 10-g sam-... 

Other ways to minimize matrix effects include improving the sample cleanup, diluting the sample, using labeled internal standards, using standard addition, or using matrix-matched standards. The last approach, however, is not permitted for enforcement methods at present by the US EPA or the US Pood and Drug Administration... 

Soil samples are extracted with methanol-water (7 3, v/v) using a Soxtec extractor. After addition of an isotopically labeled internal standard (IS) and dilution to 50 mL, the extracts are analyzed by electrospray LC/MS/MS. 

The internal standard and analyte should be resolved chromatograph-ically to baseline (except for isotoplcally labelled samples when mass discrimination or radioactive counting are used for detection), elute close together, respond to the detection system in a similar way, and be present in nearly equal concentrations. 

Quantitative analysis using FAB is not straightforward, as with all ionisation techniques that use a direct insertion probe. While the goal of the exercise is to determine the bulk concentration of the analyte in the FAB matrix, FAB is instead measuring the concentration of the analyte in the surface of the matrix. The analyte surface concentration is not only a function of bulk analyte concentration, but is also affected by such factors as temperature, pressure, ionic strength, pH, FAB matrix, and sample matrix. With FAB and FTB/LSIMS the sample signal often dies away when the matrix, rather than the sample, is consumed therefore, one cannot be sure that the ion signal obtained represents the entire sample. External standard FAB quantitation methods are of questionable accuracy, and even simple internal standard methods can be trusted only where the analyte is found in a well-controlled sample matrix or is separated from its sample matrix prior to FAB analysis. Therefore, labelled internal standards and isotope dilution methods have become the norm for FAB quantitation. 

Internal standardization involves adding a chemical standard to the sample solution so that standard and sample are effectively measured at the same time. Internal chemical standards can be either the actual analyte, an isotopically labelled analyte or a related substance. The last one is usually chosen as something expected to be absent from the sample yet expected to behave towards the measurement process in a way similar to the analyte. There are a number of different ways of using internal standards and they sometimes serve a different purpose. 

Labelled [2H3]ditallowdimethyl ammonium iodide ([2H3]DTDMAI) was synthesised to be employed as an internal standard for the quantitative determination of trace levels of DTDMAC in sewage and river water samples by using FAB-MS [117]. The synthesis of ([2H3] DTDMAI) involved the reaction of commercial ditallowmethyl amine with CD3I in 1-propanol. 

---